The Burkholderia cepacia complex (Bcc) is a group of Gram-negative bacteria that are ubiquitous in the environment and have emerged as opportunistic pathogens in immunocompromised patients. These bacteria cause significant disease in two seemingly disparate hosts: those with cystic fibrosis (CF) and those with chronic granulomatous disease (CGD). We hypothesize that there are unique and common features of Bcc that make it an opportunistic pathogen in these two life-shortening inherited diseases, in which infection is the primary cause of mortality. Surprisingly, there has not been a comprehensive study to identify the bacterial factors that are common or unique in these infections. Here we propose to identify genes important in both CF and CGD using two distinct mouse models of infection. We have already constructed a transposon mutant library encompassing approximately 60,000 random mutants of the sequenced and annotated Burkholderia cenocepacia strain J2315 using a unique mariner transposon system. Importantly, this strain has already been shown to be virulent in a CF and a CGD mouse model of infection. We will use high-throughput sequencing to identify transposon mutants missing from this library following infection in both a CF and a CGD mouse model system. This negative selection allows us to pool the mutants and use relatively few mice, rather than using large numbers of mice that would be required to screen individual Bcc mutants. Following the identification of factors required for each of these infections, we will focus on those genes important in both CF and CGD. We anticipate that genes needed for adherence and those necessary for metabolic activities in vivo will be recognized as critical for both these infections. We will construct specfic mutations in genes in these pathways and will confirm the role of these factors in the mouse models of infection. We will also determine the effect of these mutations in in vitro and ex vivo models of virulence. A thorough understanding of these processes will allow the identification of potential targets in Bcc for the development of novel antimicrobials, inhibitors, and immunotherapeutic intervention to protect these vulnerable patient populations.